The human immunodeficiency virus type 1 (HIV-1) is the retrovirus that causes the global epidemic of AIDS. The objective of the project is to explore HIV-1 glycopeptides as new immunogens for inducing neutralizing antibodies against HIV-1. Glycopeptides represent an integrated structural unity on HIV-1 envelope glycoproteins. There are grounds to consider glycopeptides as a unique epitope for HIV-1 vaccine design. For examples, certain HIV-1glycopeptides are highly conserved among HIV-1 strains and are well accessible; N-glycans can mask unwanted epitopes and redirect the immune focus; glycosylation can induce conformational epitopes; and N-glycan itself can serve as epitope of neutralizing antibodies such as the human antibody 2G12. These considerations led us to hypothesize that certain HIV-1 glycopeptides of the envelope glycoproteins constitute new neutralizing epitopes capable of eliciting broadly neutralizing antibodies against HIV-1. To test the hypothesis, the gp120 V3 domain will be chosen as a template that carries 3 highly conserved N-glycans within or adjacent to the loop. V3 domain usually raises isolate-specific antibodies. But, despite the sequence variability, V3 domain also possesses a number of conserved elements including the N-glycans, and some V3-related broadly neutralizing antibodies have been identified. The goal of the project is to turn this largely isolate-specific epitope into a more broadly neutralizing epitope for an effective immunogen. The hypothesis will be tested by pursuing three specific aims. In specific aim 1, full-size V3-domain glycopeptides of both HXB2 (X4 tropic) and Bal (R5 tropic) strains of HIV-1, together with novel, scaffold-based trivalent V3 glycopeptide immunogens, will be synthesized by a chemo-enzymatic approach. The V3-sequences will include multiple T-helper epitopes. These experiments will make available an array of homogeneous HIV-1 glycopeptides for structural and biological studies. In specific aim 2, the conformations of the V3-glycopeptides will be studied by CD, FTIR, and 2D NMR. Binding studies with V3- related neutralizing and non-neutralizing antibodies will be performed by SPR technology. These experiments will reveal how the individual N-glycans affect the conformation and antigenicity of the V3 domain. In specific aim 3, immunization studies with the synthetic immunogens will be performed in rabbits. The immunogenicity of the glycopeptide immunogens will be analyzed by ELISAs and viral neutralization assays. Theproposed research attempts to raise broadly neutralizing antibodies through novel immunogen design. Theresearch willyield important new data with direct relevance to HIV-1 vaccine development.